The long-range goal of my research is to uncover the nature of the cancer stem cells, and to design therapeutic approaches that can specifically target these cells for the treatment of breast cancers. The objective of this application is to establish the expression pattern of NS and Ngp1 in breast cancer cells, determine the growth phenotype and gene expression profile of NS- and Ngp1-expressing cells, and provide experimental evidence that demonstrates the role of NS+ cells in breast tumorigenesis. The central hypothesis is that NS and Ngp1 are preferentially expressed by the stem-like cells in breast tumors, and that these cells are responsible for the rapid growth, drug resistance, and high metastasis of malignant breast cancers. The rationale is that cancer stem cells, which are responsible for the high growth rate and drug resistance of malignant breast cancers, share common molecules such as NS and Ngp1 with somatic stem cells to drive the self-renewing machinery. Therefore, eliminating NS-expressing breast cancer cells will inhibit tumor initiation and progression. To accomplish the overall objective of this proposal, three specific aims will be pursued. The first aim is to establish the expression pattern of NS and Ngp1 in normal breast tissues and breast cancer cells. The second aim is to determine the cellular and molecular properties of those NS- and Ngp1-expressing cells by genetically marking these cells with living color proteins and purifying them by flow cytometry. The third aim is to determine the role of NS+ cells in the development, progression, and recurrence of breast tumors by evaluating the consequences of their elimination. An inducible cell ablation system will be employed to target the NS+ cells in transgenic animals. These results will provide the much-needed justification for the use of stem cell-targeting therapy in cancer treatment, and valuable information for developing new pharmacological and genetic therapies that can specifically target these cancer stem cells in order to cure advanced breast carcinomas.